ivan l. budyak forschungszentrum j ü lich, deutschland university of pittsburgh, pa, usa
DESCRIPTION
The investigations of structure and properties of membrane receptors: human EGFR and halobacterial HtrII. Ivan L. Budyak Forschungszentrum J ü lich, Deutschland University of Pittsburgh, PA, USA Московский физико-технический институт , Россия May 2006. - PowerPoint PPT PresentationTRANSCRIPT
The investigations of The investigations of structure and properties of structure and properties of
membrane receptors: membrane receptors: human EGFR and human EGFR and
halobacterial HtrIIhalobacterial HtrII
The investigations of The investigations of structure and properties of structure and properties of
membrane receptors: membrane receptors: human EGFR and human EGFR and
halobacterial HtrIIhalobacterial HtrII Ivan L. BudyakIvan L. Budyak
ForschungszentrumForschungszentrum J Jüülich, Deutschlandlich, DeutschlandUniversity of Pittsburgh, PA, USAUniversity of Pittsburgh, PA, USA
Московский физико-технический институтМосковский физико-технический институт, , РоссияРоссия
MayMay 2006 2006
PartPart 1: 1:
halobacterial transducerhalobacterial transducer II II ((HtrIIHtrII))
fromfrom Natronobacterium Natronobacterium pharaonispharaonis
PartPart 1: 1:
halobacterial transducerhalobacterial transducer II II ((HtrIIHtrII))
fromfrom Natronobacterium Natronobacterium pharaonispharaonis
Retinal-containing proteins of Retinal-containing proteins of N.pharaonisN.pharaonis
Retinal-containing proteins of Retinal-containing proteins of N.pharaonisN.pharaonis
Engelhard M. Engelhard M. et alet al. (2002), Archaeabacterial phototaxis, . (2002), Archaeabacterial phototaxis,
In In Photoreceptors and Light SignalingPhotoreceptors and Light Signaling (pp. 2-39), The Royal Society of Chemistry, UK (pp. 2-39), The Royal Society of Chemistry, UK
Two-componentTwo-component signal signal transduction system in transduction system in
N.pharaonisN.pharaonis
Two-componentTwo-component signal signal transduction system in transduction system in
N.pharaonisN.pharaonis
??
Gordeliy V.I. Gordeliy V.I. et alet al., ., NatureNature, , 419419 (2002), pp. 484-487 (2002), pp. 484-487 Oprian D.D., Oprian D.D., TIBSTIBS, , 2828 (2003), pp. 167- (2003), pp. 167-169169
transducer sequences from archaea transducer sequences from archaea including including N.pharaonisN.pharaonis are homologous are homologous to those of eubacterial chemoreceptorsto those of eubacterial chemoreceptors
both signal through the classical two-both signal through the classical two-component systemcomponent system
Structures of the cytoplasmic domains of halobacterial Structures of the cytoplasmic domains of halobacterial transducers remain unknown transducers remain unknown
open the cellsopen the cells
salting outsalting out
HIC HIC purificationpurification
gel-filtrationgel-filtration
mm1 , 1 , 2 2
33
44
55
1 1 2 2 33 44 55
Le Moual H. and Koshland D.E., Le Moual H. and Koshland D.E., J.Mol.Biol.J.Mol.Biol., , 261261 (1996), pp. (1996), pp. 568-585568-585
Tsr, T286Tsr, T286
HtrII, M234HtrII, M234
Tsr, A526Tsr, A526
HtrII, HtrII, D504D504
The cytoplasmic fragment of HtrII can be expressed in The cytoplasmic fragment of HtrII can be expressed in E.coliE.coli and purified to homogeneity and is unstructured in and purified to homogeneity and is unstructured in
solutionsolution
Design, expression, purification Design, expression, purification and and
initial characterization of HtrII-cytinitial characterization of HtrII-cyt
Design, expression, purification Design, expression, purification and and
initial characterization of HtrII-cytinitial characterization of HtrII-cyt
Structural predictions for HtrII-cytStructural predictions for HtrII-cytStructural predictions for HtrII-cytStructural predictions for HtrII-cyt
Combet C. Combet C. et al.et al., , TIBSTIBS, , 291291 (2000), pp. (2000), pp. 147-150147-150
76%76% αα-helix -helix andand 24%24% random coilrandom coil
O. Lund O. Lund et al.et al., “CPHmodels 2.0: , “CPHmodels 2.0: X3MX3M
a Computer Program to Extracta Computer Program to Extract 3D Models”, A102 abstract 3D Models”, A102 abstract
at the CASP5 conference, 2002at the CASP5 conference, 2002
Lupas A. et al., Science, 252 (1991), pp. 1162-1164
The cytoplasmic fragment of HtrII is predicted The cytoplasmic fragment of HtrII is predicted to be to be αα-helical and to form coiled coil structure-helical and to form coiled coil structure
Predictions of dynamic properties of Predictions of dynamic properties of HtrIIHtrII
Predictions of dynamic properties of Predictions of dynamic properties of HtrIIHtrII
Predictions of structural parameters are Predictions of structural parameters are contradictory contradictory
suggesting the possibility of structural transitionssuggesting the possibility of structural transitions
Romero P. et al., Proteins: Struct. Funct. Gen., 42 (2001), pp. 38-48
The cytoplasmic domain of HtrII is predicted to be The cytoplasmic domain of HtrII is predicted to be disordereddisordered
CONTIN algorithm: Van Stokkum I.H.M. CONTIN algorithm: Van Stokkum I.H.M. et al.et al., , Anal.Biochem.Anal.Biochem., , 191191 (1990), pp. (1990), pp. 110-119110-119
KClKCl NaClNaCl glycerolglycerolConformational transitionsConformational transitionsConformational transitionsConformational transitions
KCl, NaCl and glycerol induceKCl, NaCl and glycerol induceconformational transitions from mainly random coil to conformational transitions from mainly random coil to -helix-helix
CONTIN algorithm: Van Stokkum I.H.M. CONTIN algorithm: Van Stokkum I.H.M. et al.et al., , Anal.Biochem.Anal.Biochem., , 191191 (1990), pp. (1990), pp. 110-119110-119
sucrosesucrose ammonium sulfateammonium sulfate TFETFEConformational transitionsConformational transitionsConformational transitionsConformational transitions
Sucrose, ammonium sulfate and TFE also induceSucrose, ammonium sulfate and TFE also induceconformational transitions from mainly random coil to conformational transitions from mainly random coil to -helix-helix
FTIR spectroscopyFTIR spectroscopyFTIR spectroscopyFTIR spectroscopy
wavenumber, cm-1 assignment
1621-1640, 1671-1679 β-structure
1641-1647 random coil
1651-1657 α-helix
1658-1671, 1681-1690 turns and bends
1644164416541654
adapted from adapted from Stuart B. Stuart B. (1997), (1997),
Biological Biological Applications Applications of Infrared of Infrared
SpectroscopySpectroscopy, , University of University of Greenwich, Greenwich,
UKUK
FTIR indicates random coil in solution and FTIR indicates random coil in solution and -helix in dry -helix in dry filmfilm
10 mM Tris-HCl pH 9.0 in D10 mM Tris-HCl pH 9.0 in D22OO dry filmdry film
NMR spectroscopyNMR spectroscopyNMR spectroscopyNMR spectroscopy10 mM NaP pH 6.010 mM NaP pH 6.0
NMR data supportNMR data supportstructural transitions in glycerolstructural transitions in glycerol
minimal spectral dispersionminimal spectral dispersion negative het-NOE signalsnegative het-NOE signals
(data not shown)(data not shown)
10mM NaP pH 6.0 + 70% 10mM NaP pH 6.0 + 70% glycerolglycerol
peaks shifted and broadenedpeaks shifted and broadened strong Trosy effectstrong Trosy effect
Red:1H-15N HSQCGreen: Trosy-HSQC
15
15 N
chem
ical sh
ift,
ppm
N c
hem
ical sh
ift,
ppm
9.0 8.0 7.09.0 8.0 7.011H chemical shift, ppmH chemical shift, ppm
12
5 1
20
1
15
1
10
12
5 1
20
1
15
1
10
10 9 8 7 6 5 4 3 2 1 010 9 8 7 6 5 4 3 2 1 0
11H chemical shift, H chemical shift, ppmppm
8.5 8.0 7.5 7.0 6.58.5 8.0 7.5 7.0 6.5
12
5 1
20
1
15
12
5 1
20
1
15
11
01
10
15
15 N
chem
ical sh
ift,
N
chem
ical sh
ift,
ppm
ppm
AnalyticalAnalytical gel-filtration gel-filtration chromatography (AGFC)chromatography (AGFC)AnalyticalAnalytical gel-filtration gel-filtration chromatography (AGFC)chromatography (AGFC)
Abnormal retention volumesAbnormal retention volumes ofof the cytoplasmic the cytoplasmic fragmentfragment of HtrII evidence its non-globular shapeof HtrII evidence its non-globular shape;;
ammonium sulfate induces hydrophobic interactions ammonium sulfate induces hydrophobic interactions with the column up to complete retentionwith the column up to complete retention
ammonium ammonium sulfatesulfate
KClKCl andand NaClNaCl
AnalyticalAnalytical gel-filtration gel-filtration chromatography (AGFC) and chemical chromatography (AGFC) and chemical
cross-linkingcross-linking
AnalyticalAnalytical gel-filtration gel-filtration chromatography (AGFC) and chemical chromatography (AGFC) and chemical
cross-linkingcross-linking
Cross-linking data evidence HtrII-cyt dimerization in Cross-linking data evidence HtrII-cyt dimerization in 4 M KCl4 M KCl
1xPB
1xPB
SS1
xPB
S +
10
%
1xPB
S +
10
%
asas
1xPB
S +
40
%
1xPB
S +
40
%
asas
1xPB
S +
4 M
1
xPB
S +
4 M
K
Cl
KC
l
Small-angle neutron scattering Small-angle neutron scattering and atomic force microscopyand atomic force microscopy
Small-angle neutron scattering Small-angle neutron scattering and atomic force microscopyand atomic force microscopy
HtrII-cyt has characteristic size ofHtrII-cyt has characteristic size of ~180-~180-200 200 ÅÅ and elongated and elongated shape in solutionshape in solution
SANS withSANS with HtrII-HtrII-cytcyt
AFMAFM withwith HtrII-cyt in dry filmHtrII-cyt in dry film
(many thanks to Dirk Mayer, ISG-(many thanks to Dirk Mayer, ISG-2)2)
Conclusions, partConclusions, part 1 1Conclusions, partConclusions, part 1 1
The cytoplasmic The cytoplasmic domaindomain of HtrII from of HtrII from N.pharaonisN.pharaonis (HtrII-cyt) (HtrII-cyt) can be expressed in can be expressed in E.coliE.coli in soluble form and then in soluble form and then successfully purifiedsuccessfully purified
HtrII-cyt is shown to be unstructured (disordered) in common HtrII-cyt is shown to be unstructured (disordered) in common aqueous solutionsaqueous solutions
Drying and certain additives render HtrII-cyt Drying and certain additives render HtrII-cyt αα-helical with -helical with different efficacydifferent efficacy
HtrII-cyt exists in solution in monomeric form, 4 M NaCl and HtrII-cyt exists in solution in monomeric form, 4 M NaCl and KCl induce oligomerization with dimers being the most KCl induce oligomerization with dimers being the most abundant speciesabundant species
HtrII-cyt has a rod-like shape of ~200 HtrII-cyt has a rod-like shape of ~200 Å long and ~14 Å in Å long and ~14 Å in diameter for the monomeric form, and diameter for the monomeric form, and ~250 ~250 Å long and ~20 Å long and ~20 Å in diameter as a dimerÅ in diameter as a dimer
PartPart 2: 2:
human Epidermal Growth human Epidermal Growth Factor Receptor Factor Receptor ((hEGFRhEGFR))
PartPart 2: 2:
human Epidermal Growth human Epidermal Growth Factor Receptor Factor Receptor ((hEGFRhEGFR))
Epidermal Growth Factor Receptor Epidermal Growth Factor Receptor (EGFR):(EGFR):
general informationgeneral information
Epidermal Growth Factor Receptor Epidermal Growth Factor Receptor (EGFR):(EGFR):
general informationgeneral information found in a number of epithelial tissues in found in a number of epithelial tissues in
humanhuman transmembrane, 1186 a.a. long, 170 transmembrane, 1186 a.a. long, 170
kDa, 8 domainskDa, 8 domains (precursor peptide – 1212 a.a. with 26 (precursor peptide – 1212 a.a. with 26
a.a. signal sequence)a.a. signal sequence) has 3 homologous proteins in humans has 3 homologous proteins in humans
(ErbB2, ErbB3 and ErbB4) and one each (ErbB2, ErbB3 and ErbB4) and one each from from D.melanogasterD.melanogaster and and C.elegansC.elegans
posttranslationally glycosylated (20% of posttranslationally glycosylated (20% of protein mass)protein mass)
binds EGF, TGF-binds EGF, TGF-α and neuregulinsα and neuregulins exists both as monomers and dimersexists both as monomers and dimers implicated in a variety of human cancers implicated in a variety of human cancers
(e.g. mammary carcinoma, (e.g. mammary carcinoma, glioblastomas etc.)glioblastomas etc.)
Burgess A. Burgess A. et al.et al., , Mol.CellMol.Cell,,
1212 (2003), pp. 541-552 (2003), pp. 541-552
Epidermal Growth Factor Receptor Epidermal Growth Factor Receptor (EGFR):(EGFR):
extracellular and kinase domainsextracellular and kinase domains
Epidermal Growth Factor Receptor Epidermal Growth Factor Receptor (EGFR):(EGFR):
extracellular and kinase domainsextracellular and kinase domains
Burgess A. Burgess A. et al.et al., , Mol.CellMol.Cell, ,
1212 (2003), pp. 541-552 (2003), pp. 541-552
Ogiso H. Ogiso H. et al.et al., , CellCell, ,
110110 (2002), pp. 775- (2002), pp. 775-787787
residues 1-619 + EGF residues 1-619 + EGF
Stamos J. Stamos J. et al.et al., , J.Biol.Chem.J.Biol.Chem., ,
277277 (2002), pp. 46265- (2002), pp. 46265-4627246272
residues 672-998 +residues 672-998 +ATP / kinase inhibitorATP / kinase inhibitor
Epidermal Growth Factor Receptor Epidermal Growth Factor Receptor (EGFR):(EGFR):
trans- and juxtamembrane domainstrans- and juxtamembrane domains
Epidermal Growth Factor Receptor Epidermal Growth Factor Receptor (EGFR):(EGFR):
trans- and juxtamembrane domainstrans- and juxtamembrane domains
Burgess A. Burgess A. et al.et al., , Mol.CellMol.Cell, ,
1212 (2003), pp. 541-552 (2003), pp. 541-552
Rigby A. Rigby A. et al.et al., , Biochim.Biophys.ActaBiochim.Biophys.Acta, ,
13711371 (1998), pp. 241- (1998), pp. 241-253253
residues 621-654 residues 621-654
Choowongkomon K. Choowongkomon K. et alet al., ., J.Biol.Chem.J.Biol.Chem.,,280 280 (2005), pp. 24043-52(2005), pp. 24043-52
residues 645-697residues 645-697
Important information about the tj-Important information about the tj-EGFREGFR
Important information about the tj-Important information about the tj-EGFREGFR
73 amino acid residues (615-686 a.a.) (without 73 amino acid residues (615-686 a.a.) (without tags)tags)
carries N-terminal 7His-tag (HHHHHHH)carries N-terminal 7His-tag (HHHHHHH) carries C-terminal carries C-terminal StrepStrepII-tag (WSHPQFEK)II-tag (WSHPQFEK) molecular weight is about 10,152 Damolecular weight is about 10,152 Da pI is around 11.2pI is around 11.2 contains no Cys residues contains no Cys residues
L1 CR1 L2 CR2 JM Kinase CT
644
151 312 481 621 687 955 1186
Extracellular portion Intracellular portion
L1 CR1 L2 CR2 JM Kinase CT
644
151 312 481 621 687 955 1186
Extracellular portion Intracellular portion
MHHHHHHHGPKIPSIATGMVGALLLLLVVAL GIGLFMRRRH IVRKR TLRR LLQERELVEPLTPSGEAPNQALLRILKETE
tj-EGFR: why two tags?tj-EGFR: why two tags?Relation to the previous studiesRelation to the previous studies
tj-EGFR: why two tags?tj-EGFR: why two tags?Relation to the previous studiesRelation to the previous studies
Expression and Expression and purificationpurification
The results with tj-EGFR carrying ONLY The results with tj-EGFR carrying ONLY 7His-tag were unsatisfactory7His-tag were unsatisfactory
MALDI-TOFMALDI-TOF
15151010
2020
m 1 2 3 4m 1 2 3 4
1515
2020
m His-blot SDS-PAGEm His-blot SDS-PAGE
1957.0 3810.8 5664.6 7518.4 9372.2 11226.0
Mass (m/z)
0
1.1E+4
0
10
20
30
40
50
60
70
80
90
100
% In
tensi
ty
Voyager Spec #1=>BC=>NR(2.00)[BP = 5741.6, 10904]
5742.33
4819.71
4557.40
5757.75 9109.28
4564.952111.82 2874.41
5216.334533.963390.85
1957.0 3810.8 5664.6 7518.4 9372.2 11226.0
Mass (m/z)
0
1.1E+4
0
10
20
30
40
50
60
70
80
90
100
% In
tensi
ty
Voyager Spec #1=>BC=>NR(2.00)[BP = 5741.6, 10904]
5742.33
4819.71
4557.40
5757.75 9109.28
4564.952111.82 2874.41
5216.334533.963390.85
Expression of tj-EGFR in pET Expression of tj-EGFR in pET 27b+ 27b+
E.coliE.coli BL21(DE3) Codon Plus RP BL21(DE3) Codon Plus RP
Expression of tj-EGFR in pET Expression of tj-EGFR in pET 27b+ 27b+
E.coliE.coli BL21(DE3) Codon Plus RP BL21(DE3) Codon Plus RP
m – markerm – markerb – before inductionb – before inductionredred – at +37 – at +37°C / °C / blueblue – at + – at + 28°C28°C
4 – 4 hours after induction4 – 4 hours after induction16 – 16 hours after 16 – 16 hours after inductioninduction24 – 24 hours after 24 – 24 hours after inductioninduction
1515
1010
2020
2020
1010
Strep-Strep-blotblot His-blotHis-blot
m b m b 4 16 244 16 24 m b m b 4 16 244 16 24 m b m b 4 16 244 16 24 m b m b 4 16 244 16 24
The optimal expression conditions for tj-EGFR are:The optimal expression conditions for tj-EGFR are:+28+28°C, 24 hours°C, 24 hours
Purification of tj-EGFR in OG on Purification of tj-EGFR in OG on Chelating and Strep-Tactin Chelating and Strep-Tactin
SepharoseSepharose
Purification of tj-EGFR in OG on Purification of tj-EGFR in OG on Chelating and Strep-Tactin Chelating and Strep-Tactin
SepharoseSepharose
SDS-PAGE His-blot Strep-BlotSDS-PAGE His-blot Strep-Blot
open open cellscells
chelating chelating CuCu2+2+
Strep-Strep-TactinTactin
RPCRPC1515
1010
2020
tj-EGFR can be purified to tj-EGFR can be purified to homogeneityhomogeneity
MALDI-TOF analysis of tj-MALDI-TOF analysis of tj-EGFREGFR
MALDI-TOF analysis of tj-MALDI-TOF analysis of tj-EGFREGFR
999.0 3799.4 6599.8 9400.2 12200.6 15001.0
Mass (m/z)
0
7313.8
0
10
20
30
40
50
60
70
80
90
100
% I
nte
ns
ity
Voyager Spec #1=>RSM2000[BP = 10161.8, 7314]
10161.32
10217.60
5077.00
10274.985104.76
10057.235135.56 10028.83
4954.85 10013.10
4428.86
999.0 3799.4 6599.8 9400.2 12200.6 15001.0
Mass (m/z)
0
7313.8
0
10
20
30
40
50
60
70
80
90
100
% I
nte
ns
ity
Voyager Spec #1=>RSM2000[BP = 10161.8, 7314]
10161.32
10217.60
5077.00
10274.985104.76
10057.235135.56 10028.83
4954.85 10013.10
4428.86
(many thanks to Axel Niebisch, (many thanks to Axel Niebisch, IBT-1)IBT-1)
Only full-length tj-EGFR is observed: no degradation productsOnly full-length tj-EGFR is observed: no degradation products
CD spectra of tj-EGFR and CD spectra of tj-EGFR and secondary structure predictions: secondary structure predictions:
water and TFEwater and TFE
CD spectra of tj-EGFR and CD spectra of tj-EGFR and secondary structure predictions: secondary structure predictions:
water and TFEwater and TFE
α-helix β-sheet turn random
water 18% 29.5% 22.5% 30%
TFE 40% 13% 18.5% 28.5%
CD spectra of tj-EGFR and CD spectra of tj-EGFR and secondary structure predictions: secondary structure predictions:
detergentsdetergents
CD spectra of tj-EGFR and CD spectra of tj-EGFR and secondary structure predictions: secondary structure predictions:
detergentsdetergents
50 mM NaP pH 50 mM NaP pH 6.0,6.0,100 mM 100 mM detergentdetergent
α-helix β-sheet turn random
OG 21% 28% 23% 28%
SDS 21% 29% 20% 30%
DPC 32% 21% 20% 27%
DHPC 23% 25% 22% 30%
LPPG 25% 26% 19% 30%
~ 60% ~ 60% αα-helix and ~ 40% random coil-helix and ~ 40% random coil
Sequence-basedSequence-basedsecondary structure secondary structure
predictionspredictions
Sequence-basedSequence-basedsecondary structure secondary structure
predictionspredictions
MHHHHHHHGPKIPSIATGMVGALLLLLVVALGIGLFMRRRHIVRKRTLRRLLQERELVEPLTPSGEAPNQALLRILKETEWSHPQFEK
There is a discrepancy between the experimental and There is a discrepancy between the experimental and predicted secondary structure structure contentpredicted secondary structure structure content
NMR spectra of tj-EGFR in SDS and NMR spectra of tj-EGFR in SDS and DPCDPC
NMR spectra of tj-EGFR in SDS and NMR spectra of tj-EGFR in SDS and DPCDPC
2D HSQC NMR spectra look promising in terms of peak 2D HSQC NMR spectra look promising in terms of peak assignmentassignment
10 10 mM NaP pH 6.0 + mM NaP pH 6.0 + SDSSDS
10 10 mM NaP pH 6.0 + mM NaP pH 6.0 + DPCDPC
Conclusions, partConclusions, part 2 2Conclusions, partConclusions, part 2 2
The transmembrane The transmembrane + + juxtamembrane domainjuxtamembrane domain of EGFRof EGFR fromfrom HH.sapiens.sapiens ( (tjtj--EGFREGFR) can be expressed in ) can be expressed in E.coliE.coli and and then successfully purifiedthen successfully purified
tj-EGFR is prone to oligomerization/aggregationtj-EGFR is prone to oligomerization/aggregation
The secondary structure of tj-EGFR is almost independent The secondary structure of tj-EGFR is almost independent of the type of detergentof the type of detergent
The tertiary structure of tj-EGFR strongly depends on the The tertiary structure of tj-EGFR strongly depends on the type of detergent, e.g. the presence of charged headstype of detergent, e.g. the presence of charged heads
AcknowledgementsAcknowledgementsAcknowledgementsAcknowledgements
FIRST FIRST mymy BIG BOSSES BIG BOSSES:: Prof. Judith Klein-SeetharamanProf. Judith Klein-Seetharaman ( (University of PittsburghUniversity of Pittsburgh)) Prof. Georg Büldt (Forschungszentrum JProf. Georg Büldt (Forschungszentrum Jülich, IBI-2ülich, IBI-2)) Dr. Ramona Schlesinger (Forschungszentrum JDr. Ramona Schlesinger (Forschungszentrum Jülich, IBI-ülich, IBI-
22)) Dr.Dr. Valentin Gordeliy Valentin Gordeliy ((MIPTMIPT))
... and then my ... and then my NICE COLLEAGUESNICE COLLEAGUES:: Dr.Dr. Olga Mironova (HtrII-cyt, cloning & purification)Olga Mironova (HtrII-cyt, cloning & purification) Vijayalaxmi ManoharanVijayalaxmi Manoharan ( (HtrII-cyt, NMRHtrII-cyt, NMR)) Naveena YanamalaNaveena Yanamala ( (tjtj--EGFR, NMREGFR, NMR)) Prof. Joe Zaccai and Dr. Vitaliy PipichProf. Joe Zaccai and Dr. Vitaliy Pipich ( (HtrII-cyt, SANSHtrII-cyt, SANS))
What is yet to be done?What is yet to be done?– I’m not leaving you right now!– I’m not leaving you right now!
What is yet to be done?What is yet to be done?– I’m not leaving you right now!– I’m not leaving you right now!
HtrII-cyt project:HtrII-cyt project:- finalize the papers;finalize the papers;- mutagenesis (if necessary);mutagenesis (if necessary);- try to obtain diffracting crystals.try to obtain diffracting crystals.
EGFR project:EGFR project:- tj-EGFR: final CD in lipids;tj-EGFR: final CD in lipids;- tj-EGFR: cross-linking in lipids and detergents;tj-EGFR: cross-linking in lipids and detergents;- write up the paper;write up the paper;- prepare 13C, 15N sample (if necessary);prepare 13C, 15N sample (if necessary);
- N-EGFR: express in COS-1 and develop purification N-EGFR: express in COS-1 and develop purification strategy.strategy.